Microtubules are dynamic polymers that play essential roles in a variety of cellular processes such as organelle transport and chromosome segregation. The long-term objective of this proposal is to gain a better understanding of the biological role of microtubule dynamics and the ways in which these dynamic properties are regulated in the cell. Microtubule dynamics in cells is likely to be determined by the complex interplay of proteins that bind to microtubules and regulate their assembly. Three microtubule-binding proteins in the yeast S. cerevisiae, Stu2p, Bik1p and Bim1p, interact with each other in all pairwise combinations. The combinatorial effects of these proteins and the functional significance of their protein-protein interactions in regulating microtubule dynamics in vivo will be determined by making and analyzing specific mutants. In addition, the effects of these proteins, alone and in combination, on the dynamic properties of microtubules in vitro will be assayed. A fourth microtubule-binding protein, Stu1p, is found on spindle microtubules and recent findings indicate a role for Stu1p at the kinetochore. Experiments are proposed to determine if Stu1p localizes to kinetochores, the dynamics of kinetochore microtubules in cells lacking Stu1p, and the intrinsic effects of Stu1p on the dynamic properties of microtubules in vitro. Although the dynamic properties of microtubules are believed to be central to their function, the extent to which cells rely on microtubule dynamics has not been tested experimentally. Mutations in beta-tubulin that alter the dynamic properties of microtubules in S. cerevisiae will be constructed. The effect of these mutations on microtubule dynamics and function in vivo and on microtubule dynamics in vitro will be measured. The aim of this work is to understand how intrinsic changes in the dynamic properties of microtubules affect microtubule dynamics and function in the cell.